*3.3. In the Presence of Hypothetical Replication Stress, the MO Increase to Maintain Robustness in S-Phase Duration*

To predict the behavior of MO in the presence of hypothetical replication stress, we simulated two conditions considering that the S-phase duration is robust [6–8]. The first one was mild replication stress, which was characterized here by a replication rate at 2/3 of the average value from the wild type population. The second situation was harsh replication stress, with a replication rate at a 1/3 of the average value. After applying these hypothetical values in Equation (3), we estimated the MO for the first (MOMR) and second (MOHR) conditions in trypanosomatids and *S. cerevisiae*, as shown in Table 2. It is worth mentioning that we did not perform this prediction for *S. pombe* because the peculiar behavior of its cell cycle seems to contribute to a flexible (non-robust) S phase duration [25,26].

According to our prediction, the only way a cell can maintain certain robustness in the S-phase duration in the presence of mild or harsh replication stress is to increase origin activation, which was evidenced by the increase in the MO values shown in Table 2. In other words, the demand for a higher number of activated origins in the presence of replication stress characterized by a slowing of replication rate can suggest that the cell tries to maintain robustness over the S-phase duration. This predicted behavior has already been evidenced by several cell types [45–48], including trypanosomatids [8] and *S. cerevisiae* [49]. However, *S. pombe* is an exception to this, because in addition to its S-phase not being robust [25,26], in the presence of replication stress, the origin firing is inhibited [24,50]. Moreover, *S. pombe* has other features that make its cell cycle unique when compared to other organisms: the S-phase is initiated before completion of the cytokinesis of the ongoing cell cycle [26,51], the cell mass influences the duration of the S-phase [25], and the main cell cycle control point is a size control in G2 phase [51]. Altogether, these peculiarities seem to contribute to more flexibility in the S-phase duration of this yeast. Nevertheless, further studies are still needed to better understand the dynamics of the replication stress response and origin usage through the S-phase in these non-metazoan organisms.


**Table 2.** Calculation of the minimum number of origins (MO) per chromosome in the presence of mild (MOMR) and harsh (MOHR) replication stress.

<sup>1</sup> *T. cruzi*: S-phase duration = 591.6 min (current study), replication rate = 2.05 kb·min−<sup>1</sup> [23]; <sup>2</sup> *L. major*: S-phase duration = 192 min (current study), replication rate = 2.44 kb·min−<sup>1</sup> [3]; <sup>3</sup> *T. brucei*: S-phase duration = 138.6 min [8], replication rate = 3.06 kb·min−<sup>1</sup> [8]; <sup>4</sup> *S. cerevisiae*: S-phase duration = 30 min [34,35], replication rate = 1.6 kb·min−<sup>1</sup> [11].
